Cryopreserved hepatocytes are widely used in drug development as pharmaceutical companies strive to identify risk pre-clinically and shift product attrition to the earliest stages of drug discovery. Unlike immortalized cell lines, primary hepatocytes possess the full complement of metabolizing enzymes needed to represent the in vivo environment, making them cost-effective substitutes for expensive animal models in studying hepatotoxicity, drug-drug interactions, and drug metabolism. Unfortunately, supplies of fresh hepatocytes are greatly limited by donor availability. In addition, hepatocytes should be used within 5 hours of isolation, as signaling pathways that normally suppress apoptosis are disrupted when integrin-mediated binding to the extracellular matrix is eliminated. Cryopreservation alleviates the problems associated with time constraints and donor tissue availability. However, hepatocytes respond poorly when cryopreserved using current technology. Hepatocyte viability can be reduced by 20% or more, significant apoptosis is observed 24 hours after thawing, up to half of cryopreserved hepatocytes lose their ability to adhere to surfaces in vitro, and a 50% decrease in albumin and urea production is commonly observed. Together, these problems result in significant quantities of wasted hepatocytes, which ultimately increases costs for both hepatocyte suppliers and end users. In this application, we propose to improve cryopreservation outcomes by reducing apoptosis. We will establish proof-of-concept that supplementing commercially available media with hepatocyte-binding peptides will improve viability, adherence, and function of cryopreserved hepatocytes. At the conclusion of Phase 1, we expect to have a technology that can be scaled up in Phase 2 for the development of our own cryopreservation media. Ultimately, we plan to sell the cryopreservation media and to establish a primary hepatocyte cell bank that will significantly reduce the number of animal studies conducted as part of the drug development process, while simultaneously increasing the number of studies using human cells.